The accessibility of lysosomal enzymes to their substrates is affected by their subcellular compartmentalization. Routing of enzymes through the cell has been shown to be influenced by mutations occurring in the lysosomal enzyme as well as in other proteins. Due to the lack of sufficient quantities of homogeneous normal and mutant enzymes for biochemical and structural studies, we felt that it was necessary to isolate the cDNA encoding specific enzymes and by in vitro mutagenesis recreate the consequences of mutations on enzyme activity and structure. To accomplish this end, initially using Gaucher's disease as a model, we isolated and sequenced the cDNA encoding all of human glucocerebrosidase. We have described the leader sequence for this enzyme and thus have identified that portion of glucocerebrosidase that effects translocation of the enzyme to the cisternae of the endoplasmic reticulum. In order to map the other domains responsible for oligosaccharide addition and processing, substate hydrolysis, lysosomal routing and membrane association, we have synthesized oligonucleotides that are being used to mutagenize the cDNA for glucocerebrosidase. Using retroviral constructs, the cDNA was transferred to mammalian host cell lines to reconstruct Gaucher variants. This provides an in vitro cell culture model in which the compartmentalization and function of the transferred protein can be directly correlated with specific changes in the cDNA and hence protein domains. This research will provide a more rational basis for the development of therapeutic strategies using gene or gene product replacement.